Methods of screening for compounds that interact with human P2u2 purinergic receptor

a purinergic receptor and purinergic receptor technology, applied in the field of screening for compounds that interact with human p2u2 purinergic receptors, can solve the problems of difficult comparison of the relative potency of p.sub.2 -purinergic receptor agonists, the classification of sub.1 and p.sub.2 -type purinergic receptors is no longer sufficient to accurately portray this complex family of receptors, and the difficulty of sub.1 - and p.sub

Inactive Publication Date: 2000-05-16
MILLENNIUM PHARMA INC
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Benefits of technology

The constitutive expression of antisense RNA in cells has been shown to inhibit the expression of about 20 different genes in mammals and plants, and the list continually grows (Hambor, et al., J. Exp. Med. 168:1237-1245 (1988); Holt, et al., Proc. Natl. Acad. Sci. 83:4794-4798 (1986); Izant, et al., Cell 36:1007-1015 (1984); Izant, et al., Science 229:345-352 (1985) and De Benedetti, et al., Proc. Natl. Sci. 84:658-662 (1987)). Possible mechanisms for the antisense effect are the blockage of translation or prevention of splicing, both of which have been observed in vitro. Interference with splicing allows the use of intron sequences (Munroe, EMBO. J. 7:2523-2532 (1988) which should be less conserved and therefore result in greater specificity in inhibiting expression of a protein of one species but not its homologue in another species.

Problems solved by technology

As more has become known about the purinergic receptors and the wide range of physiological responses in which they play a role, the P.sub.1 - and P.sub.2 -type classifications were no longer sufficient to accurately portray this complex family of receptors.
Classification of the P.sub.2 -type purinergic receptors has been difficult because there are no published selective P.sub.2 -receptor antagonists and there are few ATP or ADP receptor-subtype specific agonists.
In addition, it has been difficult to compare the relative order of potency of P.sub.2 -purinergic receptor agonists.
Hence, this subtype has presented numerous challenges in the identification and characterization of its members.

Method used

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  • Methods of screening for compounds that interact with human P2u2 purinergic receptor

Examples

Experimental program
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example 2

Isolation of Full-length Human cDNA encoding P.sub.2U2

Insert was isolated from the PCR clone of interest (206.18), purified from an agarose gel, radiolabeled with [.alpha.-.sup.32 P]dCTP(NEN) by random-priming (Stratagene), and used to screen a DAMI cDNA library in .lambda.gt22. The library was generated using twice-selected poly-A+ mRNA (see above) and first strand cDNA synthesis was primed with an oligo-dT primer and synthesized with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Gibco / BRL). CDNA was directionally ligated into the SalI / NotI sites of the .lambda.gt22 arms and packaged (Stratagene packaging extract) and amplified in the Y1090 (r-) strain. One million clones were screened at a density of 40,000 / plate under the following conditions: duplicate nitrocellulose filters (S&S) were hybridized overnight at 42.degree. C. in a solution containing 50% deionized formamide, 5.times.SSC (sodium chloride, sodium citrate), 0.1 mg / ml heat denatured salmon sperm DNA, 0.1...

example 3

Expression of P.sub.2U2 mRNA in Various Tissues and Cell Lines

Poly-(A)+ RNA was isolated from a variety of cell lines as described above. Five .mu.g of each sample was denatured, electrophoresed on a 1.2% formaldehyde agarose gel, and transferred to nylon membrane. Blots were probed with [.alpha.-.sup.32 P]dCTP(NEN) labeled insert, as described for the library screenings, and hybridized at 42.degree. C. overnight in the following solution: 5.times.SSPE, 1.times.Denhardt's, 50% formamide, 2% sodium dodecylsulfate, 0.1 mg / ml heat denatured salmon sperm DNA. Blots were washed twice at room temperature for 15 minutes in 0.05% SDS, 2.times.SSC and then at 50.degree. C. for 30 minutes in 0.1.times.SSC, 0.1% SDS and exposed at -70.degree. C. to Kodak XAR film for 48-72 hours with an intensifying screen. Northern blots containing poly-A+ RNA from human tissues were purchased from Clontech and hybridized, washed and exposed as described above. Hybridization of RNA tissue blots with the label...

example 4

Demonstration of the Function of the Receptor in Oocytes

The native human receptor was produced in oocytes by cloning the 500 bp 5' truncation of the full-length kidney cDNA clone into the mammalian expression vector pcDNA3 (Invitrogen). Linearized DNA was used as a template for T7 polymerase (Ambion, Promega) for generation of capped in vitro transcribed mRNA following the supplier's specifications. Adult female Xenopus laevis were anesthetized in [0.015 g / l] 3-aminobenzoic acid ethyl ester for 10 minutes and 1 or 2 ovarian lobes were removed, followed by immediate suturing of the incisions. Oocytes were defolliculated at room temperature with collagenase (2 mg / ml) in Ca.sup.+2 -free medium (OR-2) for 1-2 hr. Oocytes were stored at 18.degree. C. in ND-96 (96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM HEPES (N[2-hydroxyethylpiperazine-N'[2-ethanesulfonic acid) with penicillin / streptomycin and injected with 50 nl RNA (1-2 .mu.g / ...

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Abstract

A novel subtype of the P2-purinergic receptor, referred to as the P2U2 receptor, is disclosed. This receptor is activated by four of its agonists in the following order of specificity: UTP>UDP>ADP>ATP. Nucleic acids encoding the receptor and associated screening and therapeutic methods also are disclosed.

Description

The present invention relates to a new subtype of the P.sub.2 -Purinergic receptors, which is abundantly expressed in kidney and in many cell lines of megakaryocytic or erythroleukemic origin. Referred to herein as the P.sub.2U2 receptor, this receptor is activated by ATP, ADP, UTP and UDP. The P.sub.2U2 receptor can be used as a tool to screen for agonists and antagonists that can either stimulate or block receptor activation. Such compounds have therapeutic utility in treating (1) diseases that are caused by aberrant activation of this receptor, for example over stimulation or under stimulation of the receptor and (2) diseases whose symptoms can be ameliorated by stimulating or inhibiting the activity of the P.sub.2U2 receptor.The present invention also relates to the isolated entire human gene encoding the P.sub.2U2 receptor, methods for the recombinant production of purified P.sub.2U2 receptor proteins and the proteins made by these methods, antibodies against the whole P.sub.2U...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K14/705C07K14/435G01N33/566G01N33/50A61K38/00A61K48/00C12N15/09C12N15/12C12Q1/68
CPCC07K14/705G01N33/5008G01N33/502G01N33/5044G01N33/5091G01N33/5094G01N33/566G01N33/5041A61K38/00A61K48/00G01N2500/10
Inventor CONLEY, PAMELA B.JANTZEN, HANS-MICHAEL
Owner MILLENNIUM PHARMA INC
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